P396: DYSREGULATED DNAM-1 AND KIR2DL1 EXPRESSION IN NATURAL KILLER CELLS CORRELATES WITH POOR OUTCOME IN ACUTE MYELOID LEUKEMIA
Authors: A. F O Costa, L. O Marani, I. A Lopes, L. S Binelli, P. S Scheucher, J. L S Schiavinato, M. I. A Madeira, K. B B Pagnano, B. K Duarte, A. B. F Glória, E. M Rego, F. Traina, R. Welner, L. L Figueiredo-Pontes
Journal: HemaSphere (2022)

Background: Although improvements have been made in understanding the physiopathology of Acute Myeloid Leukemia (AML), most patients still relapse leading to poor long-term prognosis and cure rates. Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms underlying the control of hematopoietic neoplasms by these cells remain unclear. Aims: We hypothesized that FLT3-ITD mutation, a molecular marker of adverse prognosis, is associated with phenotypically and functionally impaired NK cells due to imbalanced expression of inhibitory and activating receptors during AML. Methods: We analyzed the expression of DNAM-1 and KNG2D activating and NKG2A, KIR2DL1 inhibitory receptors on CD56+, CD56bright CD16- and CD56dim CD16+ NK subsets and its correlation with FLT3-ITD mutation in 70 AML patients and 12 healthy bone marrows (NBM) by flow cytometry. Seven marrows from AML patients in complete remission after induction chemotherapy (AML-CR) were used to evaluate NK cell and receptor recovery. Mann Whitney or Kruskal-Wallis tests (P < 0.05) were performed using GraphPad Prism (V8.0.2). Results: A decreased frequency of all NK cell subsets was found in AML (CD56+, P=0.03; CD56bright, P=0.04, CD56dim, P<0.01). Decreased expression of NKG2D (P<0.01) and a tendency of decreasing DNAM-1 (P=0.38) was found in CD56+ NK in AML, recovering to values closer to normal in AML-CR. Expression of NKG2D was decreased in both CD56bright and CD56dim NK subsets (P=0.03 and P<0.01), while DNAM-1 was found to be considerably decreased in CD56dim cytotoxic NK (P=0.02). FLT3 mutated patients showed even lower expression of DNAM-1 (P=0.02) and NKG2D (P=0.13) in CD56+ NK. Even though the latter was not statistically significant, we observed a decreasing pattern in these patients, that was confirmed by an important decrease of both activating receptors uniquely in the CD56dim subset (DNAM-1, P=0.03; NKG2D, P=0.05), which are known to contain the most cytotoxic activity. As for the inhibitory receptors, our cohort demonstrated a trending increase in NKG2A expression on all NK subsets, although not significant, and no difference was found in FLT3 mutated patients. Meanwhile, KIR2DL1 expression was higher in CD56+, CD56bright and CD56dim NK cells in AML (P<0.01; P=0.06 and P<0.01), while AML-CR showed frequencies comparable to healthy In FLT3-ITD mutated patients, CD56dim NK had increased expression of KIR2DL1 (P=0.03), strongly correlating with an imbalance between activation and inhibition of NK cell activity. Summary/Conclusion: Our data indicate that CD56dim NK show an exhaustion immunophenotype in FLT3-ITD mutated AML, with high expression of KIR2DL1 inhibitory receptor and low expression of both NKG2D and DNAM-1 activating receptors and that this imbalance likely leads to impaired cytotoxicity, compromised anti-leukemic activity and could be contributing to worst disease outcome, which will be confirmed through functional assays. Closing these gaps in knowledge informs the fundamental characteristics of NK dysfunction during leukemia and is of significant interest for targeting, therapeutic interventions and NK cell-mediated immunotherapy.